Clinical success of immunotherapies has been hampered by an incomplete understanding of mechanisms of immune cell homing, function, and retention in the target tissue. Our research team has taken advantage of our unique clinical resources to develop new methods, data, and insights to address these basic questions. Intraepithelial lesions (CIN2/3) caused by human papillomavirus (HPV) present an opportunity to determine how immune responses are generated and maintained in a non-sterile barrier epithelial tissue. CIN2/3 lesions are directly accessible, clinically indolent, and are associated with functionally obligate expression of viral oncoproteins E6 and E7. Although a subset undergo complete regression, peripheral blood T cell responses to viral antigens are weak, and do not correlate with disease outcome. We propose studies to identify mechanisms of immune response sequestered at the site of antigen, in CIN2/3. We have an active immunotherapy program for premalignant HPV disease, testing strategies to enhance T cell responses to viral antigens, and to enable homing and access to the female reproductive tract mucosa. We have developed a constellation of technologies to perform quantitative, tissue-based analyses of human samples obtained before and after study interventions. This proposal is based on maturing data from our two previous funding cycles, including immune therapeutic trials governed by three investigator-sponsored INDs, each of which was generated in collaboration with the NCI RAID program. Our recent preliminary data indicate that systemic therapeutic vaccination can induce a striking effector immune response in the target lesion, despite modest detectable T cell responses to vaccine antigen in the peripheral blood. Our short-term Aims are to identify the molecular signature of these tissue responses; to determine their immune therapeutic relevance; and to develop tissue signatures to predict likelihood of response either to therapeutic vaccination or to direct manipulation of the lesion microenvironment. Our long-term goal is to develop analytic algorithms for tissue-based biomarkers that will provide objective guidelines to inform development of new therapeutic strategies and to guide treatment decisions.